Spermatogonial stem cells (SSCs) are critical for maintaining spermatogenesis throughout adult life. While considerable progress has been made in our understanding of SSCs, little is known about the regulatory mechanisms that initially establish these cells in mammals. In this application, we propose to fill this gap through study of a homeobox gene-Rhox10-that we find is essential for the normal initial establishment of SSCs in the mouse testis. Rhox10 is a member of a large X-linked homeobox gene cluster that my laboratory discovered some years ago. The genes in this Rhox gene cluster are preferentially expressed in the reproductive tract, raising the possibility that they encode transcription factors critical for reproduction, but there has been little direct evidence for this As one means to test their role, we recently generated whole Rhox cluster knockout (KO) mice that lack all 33 homeobox genes in the Rhox cluster. These mice have a progressive spermatogenic decline phenotype that strongly resembles well-established mouse models harboring SSC defects. Conditional KO of the single Rhox gene that we regarded as most likely to be responsible - Rhox10 - yielded essentially the same phenotype as KO of the entire Rhox cluster. In-depth analysis of Rhox10-KO mice revealed they have a strong defect in the ability to generate SSCs during the postnatal period when SSCs are normally first established. This deficit likely results from a differentiation defect in the precursor cells that give rise to SCCs-Pro- spermatogonia (ProSG)-based on several lines of evidence, including single-cell (SC)-RNAseq analysis. Aim 1 of this proposal is to elucidate the mechanisms by which RHOX10 promotes ProSG differentiation and SSC establishment. We will first precisely define the cell subsets and developmental pathways operating during the SSC establishment period using SC-RNAseq analysis and other methods. We will then perform the same analyses on Rhox10-KO mice to define the specific roles of the RHOX10 transcription factor in ProSG differentiation and SSC establishment. As part of this analysis, we will verify candidate ProSG marker genes we have recently identified, and screen for new ones. This is critical for the field, as, to our knowledge, no well- established ProSG marker genes have previously been identified. Aim 2 of this proposal is to delineate the molecular mode of action of RHOX10 in germ cells. Our preliminary SC-RNAseq experiments have already identified candidate RHOX10-regulated genes in germ cell subsets. We will verify these candidate targets and expand our analysis to the specific germ cell subsets that we show RHOX10 acts in in vivo. RHOX10 direct targets will be identified by a battery of approaches, including ChIP-seq analysis. Functional studies will be performed to screen for target genes important for RHOX10's function in ProSG and SSC establishment. Given that little is known about this critical period of gametogenesis, these experiments will fill a large gap in the field and will provide an unprecedented view of the role o a transcription factor in vivo.